The Na-K-Cl cotransporter (NKCC) is a plasma membrane transport protein that plays a central role in cellular homeostasis. In non-polarized cells the NKCC1 isoform is involved in regulation of cell volume. In secretory epithelia, NKCC1 functions together with Cl channels, the Na pump, and K channels to bring about regulated salt movement. In the mammalian kidney another isoform, NKCC2, mediates salt absorption and is the site of action of the loop diuretic drugs furosemide and bumetanide. NKCCs are members of the cation-chloride cotransporter family, which also includes Na-Cl and K-Cl cotransporters. Previous work from this laboratory has resulted in the cloning and characterization of many of the members of this family including the NKCCs. The long term goal of this project, which focuses on NKCC1, is to understand the molecular mechanism of the cotransporter, including the structural and functional features underlying ion translocation and its regulation, and the significance of the transporter in cell and organ function. The Specific Aims of the project are: 1) We seek to further elucidate the mechanism of regulation of NKCC1, pursuing the hypothesis that phosphorylation is the key step in the process, and also examining the possible roles of dimeric assembly and vesicular trafficking. 2) We will examine the role of PASK and OSR-1 kinases in cellular regulation. Our recent data provides a convincing demonstration that PASK plays a key role in volume- and Cl-sensitive NKCC1 phosphorylation. 3) As the crucial step in understanding structure-function relationships in the cation-chloride transporters, we aim to obtain a high-resolution crystal structure of important domains of the transport protein.